Deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA") are long, threadlike macromolecules, DNA comprising a chain of deoxyribonucleotides, and RNA comprising a chain of ribonucleotides. A nucleotide consists of a nucleoside and one or more phosphate groups; a nucleoside consists of a nitrogenous base linked to a pentose sugar. Typically, the phosphate group is attached to the fifth-carbon ("C-5") hydroxyl group ("OH") of the pentose sugar; however, it can also be attached to the third-carbon hydroxyl group ("C-3 OH"). In a molecule of DNA, the pentose sugar is deoxyribose, while in a molecule of RNA, the pentose sugar is ribose. The nitrogenous bases in DNA are adenine ("A"), cytosine ("C"), guanine ("G") and thymine ("T"). These bases are the same for RNA, except that uracil ("U") replaces thymine. Accordingly, the major nucleosides of DNA, collectively referred to as "deoxynucleosides", are as follows: deoxyadenosine ("Da"); deoxycytidine ("Dc"); deoxyguanosine ("dG"); and thymidine ("T"). The corresponding ribonucleosides are designated "A"; "C"; "G"; and "U". (By convention, and because there is no corresponding thymidine ribonucleoside, deoxythymidine is typically designated as "T"; for consistency purposes, however, thymidine will be designated as "dT" throughout this disclosure).
The sequence of the nitrogenous bases of the DNA and RNA molecule encodes the genetic information contained in the molecule. The sugar and phosphate groups of a DNA or RNA molecule perform a structural role, forming the backbone of the molecule. Specifically, the sugar moiety of each nucleotide is linked to the sugar moiety of the adjacent nucleotide such that the 3'-hydroxyl of the pentose sugar of one nucleotide is linked to the 5'-hydroxyl of the pentose sugar of the adjacent nucleotide. The linkage between the two pentose sugars is typically via a phosphodiester bond. Based upon this linkage protocol, one end ("terminus") of the nucleotide chain has a 5'-terminus (e.g. hydroxyl, triphosphate, etc.), and the other end has a 3'-hydroxyl group. By convention, the base sequence of a nucleotide chain is written in a 5' to 3' direction, i.e., 5'-ATCG-3', or, simply ATCG.
While DNA and RNA are produced internally by living animals, DNA and RNA can be chemically synthesized such that synthetic strands of DNA and RNA can be rapidly and effectively produced. These strands are typically referred to as "synthetic oligonucleotides" or "oligonucleotides". A widely utilized chemical procedure for the synthesis of oligonucleotides is referred to as the "phosphoramidite methodology". See, e.g., U.S. Pat. No. 4,415,732; McBride, L. and Caruthers, M. Tetrahedron Letters, 24:245-248 (1983); and Sinha, N. et al. Nucleic Acid Res; 17:4539-4557 (1984), which are all incorporated herein by reference. Commercially available oligonucleotide synthesizers based upon the phosphoramidite methodology include, e.g., the Biosearch 8750.TM. and ABI 380B.TM., 392.TM. and 394.TM. DNA synthesizers.
The importance of chemically synthesized oligonucleotides is principally due to the wide variety of applications to which oligonucleotides can be directed. For example, oligonucleotides can be utilized in biological studies involving genetic engineering, recombinant DNA techniques, antisense DNA, detection of genomic DNA, probing DNA and RNA from various systems, detection of protein-DNA complexes, detection of site directed mutagenesis, primers for DNA and RNA synthesis, primers for amplification techniques such as the polymerase chain reaction, ligase chain reaction, etc, templates, linkers, and molecular interaction studies. Of increasing importance is the synthesis of antisense DNA, or oligodeoxyribonucleoside phosphorothioates, as a class of therapeutic agents and efforts to improve their preparation are increasing.
The primary structures of DNA and RNA molecules can be depicted as follows: ##STR1##
The key step in nucleic acid synthesis is the specific and sequential formation of internucleotide phosphate linkages between a 5'-OH group of one nucleotide and a 3'-OH group of another nucleotide. Accordingly, in the typical synthesis of oligonucleotides, the phosphate group of an "incoming" nucleotide is combined with the 5'-OH group of another nucleotide (i.e. the 5'-OH group is "phosphorylated" or "phosphitylated"). These groups must be capable of actively participating in the synthesis of the oligonucleotide. Thus, the 5'-OH groups are modified (typically with a dimethoxy trityl ("DMT") group) such that an investigator can introduce two such nucleotides into a reaction chamber and adjust the conditions therein so that the two nucleotides are properly combined; by a series of successive such additions, a growing oligonucleotide having a defined sequence can be accurately generated.
The four bases of the nucleosides, adenine, thymine (uracil in the case of RNA), guanosine and cytosine, include moieties which are chemically reactive (e.g., exocyclic amino groups). These groups, unlike the 3'-OH and 5'-OH groups, must be "temporarily" protected, i.e. the protecting groups must be capable of blocking any reactive sites on the base until after the oligonucleotide synthesis is completed; after such synthesis is completed, these groups must also be capable of being removed from the bases such that the biological activity of the oligonucleotide is not affected.
The principal reason for temporarily protecting the base is that in the absence of such protecting groups, the exocyclic amino groups ("NH.sub.2 ") of the bases can compete for binding to the 5'-OH group. If such a reaction takes place, the resulting product will not be useful. Accordingly, these protecting groups are important in reducing the occurrence of "side product formation" i.e. the formation of chemically similar, but unwanted, materials. Cytidine is particularly susceptible to side product formation during oligonucleotide cleavage and deprotection (i.e. the processes of removing an oligonucleotide from a solid support and removing such protecting groups, respectively). The most widely used protecting groups used in conjunction with the phosphoramidite methodologies for oligonucleotide synthesis are benzoyl for A and C, and isobutyryl for G and C, (thymine, which does not have an amino group, does not ordinarily require a protecting group). By convention, benzoyl is designated "bz", and isobutyryl is designated "ibu", such that the deoxynucleosides protected therewith are typically designated as follows: Da.sup.bz ; Dc.sup.bz ; Dc.sup.ibu ; and Dg.sup.ibu.
Benzoyl and isobutyryl have the following structures: ##STR2## Beneficially, these protecting groups can be removed from the oligonucleotide with ammonia (i.e., "deprotected"). Additionally, ammonia can be used to remove oligonucleotides from the solid support material from which they were synthesized (i.e., "cleavage"). Advantageously, ammonia can be used as a cleavage/deprotection reagent with limited side product formation.
A practical concern exists, however, with respect to the use of ammonia as a cleavage and deprotection reagent. Ammonia requires a (relatively) long time period to complete the cleavage and deprotection process. On average, 6 minutes is required for the chemical synthesis of each nucleoside to a growing oligonucleotide; thus, for an average oligonucleotide of about 21 nucleotides (referred to as a "21-mer"), one can expect that the synthesis will require about 2 hours, using commercially available DNA synthesizers. However, approximately 24 hours (room temperature) to 6 hours (55.degree. C.) are required for cleavage and deprotection of the oligonucleotide using ammonia. Clearly, more time is required for the final steps of cleavage and deprotection than the synthesis itself. As such, an ongoing need has existed for cleavage and deprotection reagents which can complete these steps within the same approximate order of magnitude as the synthesis itself. Such reagents are disclosed in the related application referenced above, which is incorporated herein by reference.
Broadly, such reagents comprise at least one straight chain alkylamine comprising from 1 to about 10 carbon atoms (such an alkylamine can be represented as follows: --NH.sub.2 (CH.sub.2).sub.0-10 --CH.sub.3). In a particularly preferred embodiment of the reagent disclosed in the above-referenced application, a reagent comprising methylamine and t-butylamine can be utilized to cleave and deprotect oligonucleotides in less than about 90 minutes at room temperature, and less than about 10 minutes at about 65.degree. C.
It was observed that when these reagents are used in conjunction with oligonucleotides comprising dC.sup.ibu or dC.sup.bz, the formation of an unwanted side product, N-methylcytidine, could occur. With respect to dC.sup.bz, approximately 10% of the cytidines within the oligonucleotides were N-methylcytidine. Thus, while on the one hand a cleavage/deprotection reagent which could rapidly accomplish these tasks had been discovered, on the other hand such reagent, when used in conjunction with the so called "traditional" bz and ibu protecting groups for the base cytidine, led to cytidine side product formation.
What is needed, then, are protecting groups useful in oligonucleotide synthesis which do not have such deleterious side effects.